Sasha Celada
Sasha Celada
Sasha Celada
Helios Scholar
School: Grand Canyon University
Hometown: Moline, Illinois
Mentored by: Muhammed Murtaza, Ph.D.

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The Characterization of Cell-free DNA using Shallow Whole Genome Sequencing

In different types of cancer, circulating tumor DNA (ctDNA) in blood-derived plasma has emerged as a potential biomarker for tumor genotyping, treatment monitoring, and early tumor detection. Depending on pre-analytical factors, the quality of the plasma sample can vary. Specifically, longer cellular DNA accounts for approximately 10% of a plasma sample. Previously, a digital droplet PCR (ddPCR) assay was optimized to quantify the fraction between amplifiable copies of short cell-free DNA (cfDNA) and long cellular DNA. However, shallow whole genome sequencing (sWGS) implementation can reveal changes in copy numbers that can be used to derive a tumor fraction. Libraries were prepared using the SMARTer ThruPLEX Plasma-Seq Kit with sheared genomic DNA and extracted cfDNA from plasma. Libraries were prepared using different input volumes and cycle numbers. The size and concentration of individual libraries were assessed using Tapestation and Qubit. In preliminary results, both the sheared genomic DNA and the extracted cfDNA seem to follow the expected pattern of a plateau. The sheared genomic DNA has a large fraction of short fragments, while extracted cfDNA has a significantly smaller fraction of short fragments; therefore, less fragments were available for conversion. The sheared genomic DNA produced a higher concentration of libraries than the plasma cfDNA. We believe that the fraction of long fragments affects the overall library preparation kit performance. Future directions should include: library preparation with higher quality plasma samples and more sheared genomic DNA. After sequencing the libraries, the characterization of the cfDNA through sWGS can be compared to the previous method of ddPCR. 

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